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Compositions and methods for genetic modification and targeting

A genetic modification, mammalian technology, applied in the field of organs and non-human mammals, cells, and tissues, which can solve the problems of expensive and time-consuming

Pending Publication Date: 2021-10-19
HANGZHOU QIHAN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, conferring or enhancing resistance to viral infection through gene editing can be costly, time-consuming and sometimes ineffective

Method used

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  • Compositions and methods for genetic modification and targeting
  • Compositions and methods for genetic modification and targeting
  • Compositions and methods for genetic modification and targeting

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0199] Example 1. Genetically modified pigs exhibit enhanced resistance to ASFV

[0200] Genetically modified pigs comprising knockouts of three endogenous genes (CMAH, GGTA1 and B4GALNT2) were examined for resistance to ASFV. Porcine alveolar macrophages were isolated from genetically modified triple knockout pigs (3KO group) and unmodified controls (WT group). Porcine alveolar macrophages were seeded on polylysine-coated plates and challenged with ASFV infection at various dilutions. After ASFV infection, supernatants and cell pellets were collected and examined for ASFV viral replication. Figure 1A Shown in the use of reduced virus titer (10 -2 ) on day 2, ASFV replication was significantly reduced in the 3KO group compared to the WT control group. ASFV virus copy numbers in supernatants and alveolar macrophage pellets were measured by qPCR. Figure 1B It was shown that the supernatant obtained from the 3KO group exhibited lower ASFV virus copy number within 72 hours. ...

Embodiment 2

[0201] Example 2. Genetic modification of endogenous RELA gene

[0202] To enhance resistance to viral infection by ASFV, the endogenous RELA gene was genetically modified in pigs. Figure 2A The top panel of , shows the locus (exon 13) into which the modified RELA allele was introduced by CRISPR / Cas9-directed homologous recombination. The modified RELA allele corresponds to the ASFV-resistant warthog RELA allele and is flanked at the 5' and 3' ends by 1.5 kb homology arms (e.g. the first 1.5 kb homology arm followed by the modified allele gene, followed by a second 1.5 kb homology arm). Figure 2A The lower panel of , shows the guide nucleic acid (gRNA) targeting and binding sites for gRNA 1 and gRNA 2. Change the gRNA target site in the donor plasmid to a synonymous codon. Figure 2B Screening of candidate clones with biallelic modified RELA knock-ins is shown. The PCR product shown in the agarose gel corresponds to the presence of the knock-in RELA allele. Figure 2C S...

Embodiment 3

[0203] Example 3. Targeting multiple regions of the ASFV viral genome for cleavage and degradation

[0204] To effectively combat viral infections, guide nucleic acids (gRNAs) were designed for multiple targeting of viral genomes. Such multiplexing reduces the likelihood of the viral genome escaping cleavage due to mutations in the viral genome. The gRNA is also designed to avoid targeting and binding of the host cell's genomic sequence. Other design criteria included targeting ASFV coding and conserved regions. The gRNA was designed to be recognized by Cas9 from Streptococcus pyogenes, which recognized the PAM sequence 5'-NGG-3' (where "N" can be any nucleotide base) as part of the gRNA. Targets with a GC content between 20% and 80% have high priority; avoid homopolymers of U nucleotides; and avoid off-target cleavage with up to two mismatches with the host cell's genome.

[0205] gRNAs were designed to target the sequences shown in Table 3. SEQ ID NO: 6 is the nucleic ac...

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Abstract

Described herein are compositions and methods for modifying and targeting genes. Also described herein are compositions and methods for modifying and targeting genes in cells or non-human mammals.

Description

[0001] Incorporate by reference [0002] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Background technique [0003] Treating and preventing viral infections and diseases is one of the most pressing challenges of modern medicine. The damage from viral infection and disease ranges widely. For example, viral infections and diseases of crops and livestock cause billions of dollars in losses each year. [0004] Genetic modification has been exploited to combat viral infections. Genetic modification may involve the manipulation of genes, including the addition, deletion, or replacement of a target gene, or a portion thereof, at single or multiple loci within the genome. This approach confers or increases resistance to viral infection by geneticall...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/113C12N15/90C12N9/22A01K67/027
CPCC07K14/70596C12N15/907A01K67/0276C12N15/1131C12N9/22A01K2227/108A01K2267/02A01K2217/075C12N2310/20A01K67/0275A01K2217/072C12N15/113C12N2320/11C12N15/11
Inventor 杨璐菡高杨滨
Owner HANGZHOU QIHAN BIOTECHNOLOGY CO LTD
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